Mitochondrial reactive oxygen species (ROS) and oxidative damage are associated with a range of age-related human pathologies. It is also likely that mitochondrial ROS generation is a factor in stress response and signal transduction pathways. However, current methods for measuring and influencing mitochondrial ROS production in vivo often lack the desired specificity. To help elucidate the potential role of mitochondrial ROS production in ageing, we have developed a range of mitochondria-targeted ROS probes that may be useful in vivo. This was achieved by covalently attaching a lipophilic cation to a ROS-reactive moiety causing its membrane potential-dependent accumulation within mitochondria. Mitochondria-targeted molecules developed so far include antioxidants that detoxify mitochondrial ROS, probes that react with mitochondrial ROS, and reagents that specifically label mitochondrial protein thiols. Here, we outline how the formation and consequences of mitochondrial ROS production can be investigated using these probes.